CN111718974B - Application of mung bean peptide in preparation of medicine for promoting lead excretion, active peptide and application thereof - Google Patents
Application of mung bean peptide in preparation of medicine for promoting lead excretion, active peptide and application thereof Download PDFInfo
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Abstract
The invention provides application of mung bean peptide in preparing a medicine for promoting lead excretion, active peptide and application thereof, belonging to the technical field of active peptide, wherein the preparation method of the mung bean peptide comprises the following steps: mixing the mung bean protein isolate with water, and treating the mixture at 50-55 ℃ for 50-70 min to obtain a treated substance; adjusting the pH value of the treated substance to 8.5, mixing with alkaline protease for enzymolysis, adjusting the pH value of the zymolyte to 5, inactivating the enzyme of the adjusted substance to obtain an enzyme-inactivated substance, adsorbing the enzyme-inactivated substance for 50-70 min by activated carbon at 55-60 ℃, sequentially passing through a 30000Da ceramic membrane and a 1KD organic membrane, and drying to obtain the mung bean peptide. The mung bean peptide and the active peptide provided by the invention have the effects of promoting lead excretion, promoting motor nerve injury regeneration caused by lead poisoning and protecting axon injury caused by lead poisoning.
Description
Technical Field
The invention belongs to the technical field of polypeptide, and particularly relates to application of mung bean peptide in preparation of a medicine for promoting lead excretion, active peptide and application thereof.
Background
Lead pollutants are widely present in the living environment of people, and when lead poisoning occurs, the nervous, cardiovascular, skeletal, reproductive and immune systems of the human body are damaged. And cause malignant diseases of stomach, intestine, liver, kidney, etc. The main measures for preventing lead poisoning are far away from lead pollution areas and change the existing dietary habits of people. At present, the treatment of lead toxicity is mainly divided into two main categories, clinical treatment and health care treatment. The development and utilization of natural special medical health food have more important significance for people who accumulate a certain amount of lead in vivo and do not meet the clinical poisoning treatment standard.
At present, the variety of lead removal drugs meeting these conditions and widely used in clinical applications is relatively rare. Therapeutic agents are mainly sulfhydryl competitive antidotes and ovocarboxy metal chelators. The injection and oral treatment methods mainly aim at the clinical treatment of lead poisoning. The medicines have no difference in removing lead and other heavy metal ions in body fluid and histiocyte, and also have the characteristics of hepatotoxicity and kidney toxicity and inconvenient administration.
As a preventive and therapeutic drug, there are also simple Chinese medicine prescriptions and nutritious health-care drinks, etc. Although having a certain curative effect, the traditional Chinese medicine composition has the defects of slow response, long period, high relapse rate after stopping taking the medicine, insufficient lead-removing purpose and the like.
Disclosure of Invention
In view of the above, the present invention aims to provide an application of mung bean peptide in the preparation of a drug for promoting lead excretion, an active peptide and an application thereof, wherein the active peptide has a lead excretion promoting effect on lead.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides an application of mung bean peptide in preparing a medicine for promoting lead excretion, wherein the preparation method of the mung bean peptide comprises the following steps:
1) mixing the mung bean protein isolate with water to obtain a mixture, and treating the mixture at 50-55 ℃ for 50-70 min to obtain a treated substance;
2) adjusting the pH value of the treated matter obtained in the step 1) to 8.5, mixing the treated matter with alkaline protease, performing enzymolysis for 2.5-3.5 hours at the temperature of 55-65 ℃ to obtain an zymolyte, and adjusting the pH value of the zymolyte to 5 to obtain a regulator;
3) inactivating enzyme of the regulator obtained in the step 2) to obtain an enzyme-inactivated substance, adsorbing the enzyme-inactivated substance for 50-70 min by using active carbon at 55-60 ℃, sequentially passing through a 30000Da ceramic membrane and a 1KD organic membrane, and drying to obtain the mung bean peptide.
Preferably, the ratio of the mass of the mung bean protein isolate in the step 1) to the volume of water is 1000kg: 10000L.
Preferably, the mass ratio of the treated substance in the step 2) to the alkaline protease is 100: 3.
The invention also provides application of the mung bean peptide in preparing a medicine for promoting motor nerve injury regeneration caused by lead poisoning.
The invention also provides application of the mung bean peptide in preparing a medicine for protecting axonal injury caused by lead poisoning.
The invention also provides an active peptide, and the amino acid sequence of the active peptide is shown as SEQ ID No. 1.
Preferably, the preparation of the active peptide comprises: mixing the mung bean peptide of claim 1 with water to obtain a mixed solution, separating and purifying the mixed solution by a Sephadex G-50 chromatographic column, and collecting components with retention time of 18.5min to obtain the active peptide.
The invention also provides application of the active peptide in the technical scheme in preparation of a medicine for promoting lead excretion.
The invention also provides application of the active peptide in the technical scheme in preparing a medicine for promoting motor nerve injury regeneration caused by lead poisoning.
The invention also provides application of the active peptide in the technical scheme in preparing a medicine for protecting axonal damage caused by lead poisoning.
The invention provides an application of mung bean peptide in preparing a medicine for promoting lead excretion, wherein the preparation method of the mung bean peptide comprises the following steps: 1) mixing the mung bean protein isolate with water to obtain a mixture, and treating the mixture at 50-55 ℃ for 50-70 min to obtain a treated substance; 2) adjusting the pH value of the treated matter obtained in the step 1) to 8.5, mixing the treated matter with alkaline protease, performing enzymolysis for 2.5-3.5 hours at the temperature of 55-65 ℃ to obtain an zymolyte, and adjusting the pH value of the zymolyte to 5 to obtain a regulator; 3) inactivating enzyme of the regulator obtained in the step 2) to obtain an enzyme-inactivated substance, adsorbing the enzyme-inactivated substance for 50-70 min by using active carbon at 55-60 ℃, sequentially passing through a 30000Da ceramic membrane and a 1KD organic membrane, and drying to obtain the mung bean peptide.
The results of the embodiments of the present invention show that: the mung bean peptide and the active peptide provided by the invention have the effects of promoting lead excretion, promoting motor nerve injury regeneration caused by lead poisoning and protecting axon injury caused by lead poisoning.
Drawings
FIG. 1 shows the lead content in zebrafish in treated group compared to model control group, p <0.05
FIG. 2 is a graphical representation of the regeneration promoting effect of a zebrafish peripheral motor nerve injury of component one and component two with red dashed lines representing peripheral motor nerve length;
FIG. 3 shows the peripheral motor nerve lengths of zebrafish component one and component two, compared to model control, p < 0.05;
figure 4 shows the regeneration promoting effect of zebrafish peripheral motor nerve injury with component one and component two, compared to model control group,. p < 0.05;
FIG. 5 is a graphical representation of the protective effect of the first and second components on axonal injury of zebrafish, with red fluorescent markers within the yellow dashed line representing the axonal bundles in the brain;
FIG. 6 shows the effect of zebrafish component one and component two on the fluorescence intensity of the axons of the head, compared to the model control group,. p <0.05,. p <0.01,. p.p.ltoreq.0.001;
FIG. 7 shows the protective effect of zebrafish component one and component two on axonal injury, p <0.05, p <0.01, p < 0.001.
FIG. 8 shows the secondary mass spectrum of liquid chromatography-QEX mass spectrometry of mung bean peptide (Tyr-His-Asp-Ile-Phe-Met-Pro-Tyr).
Detailed Description
The invention provides an application of mung bean peptide in preparing a medicine for promoting lead excretion, wherein the preparation method of the mung bean peptide comprises the following steps:
1) mixing the mung bean protein isolate with water to obtain a mixture, and treating the mixture at 50-55 ℃ for 50-70 min to obtain a treated substance;
2) adjusting the pH value of the treated matter obtained in the step 1) to 8.5, mixing the treated matter with alkaline protease, performing enzymolysis for 2.5-3.5 hours at the temperature of 55-65 ℃ to obtain an zymolyte, and adjusting the pH value of the zymolyte to 5 to obtain a regulator;
3) inactivating enzyme of the regulator obtained in the step 2) to obtain an enzyme-inactivated substance, adsorbing the enzyme-inactivated substance for 50-70 min by using active carbon at 55-60 ℃, sequentially passing through a 30000Da ceramic membrane and a 1KD organic membrane, and drying to obtain the mung bean peptide.
Mixing the mung bean protein isolate with water to obtain a mixture, and treating the mixture at 50-55 ℃ for 50-70 min to obtain a treated substance.
In the invention, the ratio of the mass of the mung bean protein isolate to the volume of water is preferably 1000kg: 10000L. In the present invention, the water is preferably deionized water.
The mixture is treated at 50-55 ℃ for 50-70 min, preferably at 50-55 ℃ for 60 min.
The pH value of the treated substance obtained by adjusting is 8.5, the treated substance is mixed with alkaline protease and then is subjected to enzymolysis for 2.5-3.5 hours at the temperature of 55-65 ℃ to obtain an zymolyte, and the pH value of the zymolyte is adjusted to be 5 to obtain an adjusted substance.
In the present invention, the pH of the treated product is preferably adjusted by using a 1M sodium hydroxide solution. In the present invention, the mass ratio of the treated product to the alkaline protease is preferably 100: 3. In the present invention, the time of the enzymolysis is preferably 3h, and the temperature of the enzymolysis is preferably 60 ℃. The pH of the substrate is preferably adjusted using a 1M hydrochloric acid solution.
The method comprises the steps of inactivating enzyme of an obtained regulator to obtain an enzyme-inactivated substance, adsorbing the enzyme-inactivated substance for 50-70 min by using active carbon at the temperature of 55-60 ℃, sequentially passing through a 30000Da ceramic membrane and a 1KD organic membrane, and drying to obtain the mung bean peptide.
In the present invention, the temperature of the enzyme deactivation is preferably 110 ℃, and the time of the enzyme deactivation is preferably 5 min. In the present invention, the drying is preferably spray drying.
The invention also provides application of the mung bean peptide in preparing a medicine for promoting motor nerve injury regeneration caused by lead poisoning.
The invention also provides application of the mung bean peptide in preparing a medicine for protecting axonal injury caused by lead poisoning.
The dosage form of the medicine is not particularly limited, and the mung bean peptide can be prepared into a medically acceptable dosage form.
The invention also provides an active peptide, the amino acid sequence of which is shown as SEQ ID No.1 and specifically comprises the following components:
YHDIFMPY。
in the present invention, the preparation of the active peptide preferably comprises: mixing the mung bean peptide and water to obtain a mixed solution, separating and purifying the mixed solution by a Sephadex G-50 chromatographic column, and collecting components with retention time of 18.5min to obtain the active peptide.
In the present invention, the concentration of the active peptide in the mixture is preferably 120 mg/ml. In the present invention, the Sephadex G-50 column is 3X 100cm in size. In the present invention, the parameters of the separation and purification are preferably: the mobile phase was 10% methanol, the flow rate was 1ml/min, and the absorbance at 214nm of the eluate was measured.
The invention also provides application of the active peptide in the technical scheme in preparation of a medicine for promoting lead excretion.
The invention also provides application of the active peptide in the technical scheme in preparing a medicine for promoting motor nerve injury regeneration caused by lead poisoning.
The invention also provides application of the active peptide in the technical scheme in preparing a medicine for protecting axonal damage caused by lead poisoning.
The dosage form of the medicine is not particularly limited, and the active peptide can be prepared into a pharmaceutically acceptable dosage form.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The production method of the mung bean peptide comprises the following steps:
step one, 1000kg of mung bean protein isolate is taken for size mixing, 10000L of deionized water is added, and the concentration of a substrate is 10%.
Step two, uniformly stirring the mung bean protein isolate solution obtained in the step one, heating to 55 ℃, and preserving heat for 1 h.
And step three, adjusting the pH of the solution obtained in the step two to 8.5 by using 1MNaOH, and heating to 60 ℃.
And step four, adding 3 percent of alkaline protease (30Kg) by mass into the solution obtained in the step three, adding 1M HCl after enzymolysis for 3 hours, and adjusting the pH value to 5.
And step five, heating the solution in the step four to 110 ℃, and keeping for 5 min.
And step six, cooling the enzyme deactivation solution obtained in the step five to 55-60 ℃, adding activated carbon, keeping the temperature for 1 hour, passing through a 30000Da ceramic membrane, passing through a 1Kd organic membrane, and performing spray drying to obtain the mung bean peptide.
Example 2
Separation and purification of mung bean peptide
Step one, the mung bean peptide produced in the example 1 is dissolved in water to prepare 120mg/mL, separation and purification are carried out by a Sephadex G-50 chromatographic column (3 x 100cm), the mobile phase is 10% methanol, the flow rate is 1mL/min, the absorbance of the eluent at 214nm is measured, the fractions are collected according to chromatographic peaks, and the fractions are frozen and dried into powder by a vacuum drier after being concentrated.
And step two, verifying the lead removing effect of different components by using the zebra fish lead poisoning model of the different components obtained in the step one, and finding that a chromatographic peak with the retention time of 18.5min has a good lead removing promoting effect. The component is further separated and purified by high performance liquid chromatography, and then the structure identification is carried out.
And step three, identifying the purity and the structure: and (3) identifying the purity and the structure of the different components obtained in the step one by adopting a nano-liter liquid chromatography-Q extraction mass spectrum combined system.
Chromatographic conditions are as follows:
(1) the mobile phase A comprises 100% of purified water and 0.1% of formic acid; phase B is 100% acetonitrile + 0.1% formic acid;
(2) flow rate of mobile phase 300nl/min
(3) Sample size 1. mu.L of supernatant
(4) Mobile phase gradient program:
table 1 mobile phase gradient procedure
| Time (minutes) | 0 | 2.0 | 36.0 | 38.0 | 41.0 | 42.0 | 45.0 |
| A(％) | 97 | 97 | 63 | 10 | 10 | 97 | 97 |
| B(％) | 3 | 3 | 37 | 90 | 90 | 3 | 3 |
The single-chain polypeptide with the structure of YHDIFMPY (Tyr-His-Asp-Ile-Phe-Met-Pro-Tyr) is identified by using nano-upgrading liquid chromatography-Q EXACTIVE mass spectrum combination. The content of the polypeptide single chain in the mung bean peptide is 7.9 percent by the liquid chromatography-mass spectrometry technology, and the content of the polypeptide single chain in the mung bean peptide reaches 89 percent after the polypeptide single chain is separated and purified by a Sephadex G-50 chromatographic column. Therefore, the mung bean peptide obtained after the separation and purification by the Sephadex G-50 chromatographic column in the example 2 is named as a component I; the 18.5 chromatographic peak component separated and purified by the Sephadex G-50 chromatographic column in the example 2 is identified to have a sequence of YHDIFMPY (Tyr-His-Asp-Ile-Phe-Met-Pro-Tyr) by nano-liter liquid chromatography-Q EXACTIVE mass spectrometry, and the peptide is synthesized by Shanghai Qiaozhao biological company and named as component two (namely, active peptide).
Example 3
Zebra fish lead poisoning model evaluation component I and component II lead excretion promoting effect
Wild type AB strain zebrafish 2 hours (2hpf) after fertilization of 240 fish were randomly selected in six well plates with 30 tails per well and a 3mL per well capacity, for a total of 6 parallel groups. The concentration of the first component 27.8, 83.3 and 250 mug/mL and the concentration of the second component 27.8, 83.3 and 250 mug/mL are dissolved in water, the concentration of the sodium calcium edetate is 125 ng/tail is injected into the positive control drug, and a normal control group (zebra fish treated by water for fish culture) and a model control group are arranged at the same time. The method comprises the steps of dissolving lead acetate in water to establish a lead poisoning model, collecting zebra fish samples (cleaning liquid medicine and sucking dry fish culture water) by using an EP (inductively coupled plasma) tube after 70 hours of administration, taking 1 sample per 60 zebra fish, detecting the lead content of each zebra fish sample by using ICP-MS (inductively coupled plasma-Mass Spectrometry), converting the ratio of the lead content to the wet weight of the zebra fish to calculate the lead content (C) in the zebra fish, and evaluating the lead excretion promoting effect of pure peptide, namely active peptide () and mung bean peptide compound according to the statistical significance of the lead content. The statistical treatment results are expressed as mean ± SE; the calculation formula of the test product lead excretion promoting effect is as follows:
statistical analysis was performed using independent sample T test, with p <0.05 indicating significant differences, providing representative experimental profiles.
The results show that: the lead content (9.01 mu g/g) in the zebra fish of the lead poisoning model control group is obviously higher than that of the normal control group (3.11 mu g/g), and the successful construction of the model is prompted (p is less than 0.01). The positive control drug calcium disodium edetate 125 ng/tail body lead content (8.38 mu g/g) is compared with a model control group (9.01 mu g/g) by p >0.05, and the lead excretion promoting effect is 7 percent, which shows that the calcium disodium edetate has a certain lead excretion promoting effect trend under the experimental dosage condition.
The lead content in the zebra fish with the first component of 27.8, 83.3 and 250 mu g/mL concentration is 8.69, 8.28 and 6.43 mu g/g, compared with a model control group in pairs, p is more than 0.05 and less than 0.05, and the lead excretion promoting effect is 3.6%, 8% and 29%; the lead contents in the zebra fish with the concentrations of the second components of 27.8, 83.3 and 250 mug/mL are respectively 8.39, 7.95 and 6.31 mug/g, compared with a model control group in pairs, p is more than 0.05 and p is less than 0.05, and the lead excretion promoting effect is respectively 6.9%, 11.8% and 30%. The compound of the first component and the second component has obvious lead excretion promoting effect.
TABLE 2 Effect of component one and component two on the lead content in Zebra fish
Note: each experimental group was tested on 3 replicates and 60 zebra fish were collected for each replicate; p <0.05 compared to model control group
Example 4
Evaluation of regeneration promoting action of component one and component two on motor nerve injury caused by lead poisoning
The 240-tail 2hpf transgenic motor neuron fluorescent zebra fish NBT strain is randomly selected to be placed in a six-well plate, 30 tails are placed in each well, the volume of each well is 3mL, 27.8, 83.3 and 250 mu g/mL of a first component and 27.8, 83.3 and 250 mu g/mL of a second component are supplied to the NBT strain through water dissolving, 125 ng/tail of calcium disodium edetate is supplied to the NBT strain through positive control medicine injection, and a normal control group (water-treated zebra fish for fish culture) and a model control group are set at the same time. The lead poisoning model is established by adding lead acetate into water, after 70 hours of administration, photographing is carried out under a fluorescence microscope, the average value (L) of the lengths of peripheral motor nerves of the area where three somites above the cloaca of the zebra fish are located is collected, and the motor nerve regeneration promoting effect of the pure peptide and mung bean peptide compound is evaluated according to the statistical significance of the lengths of the motor nerves. The regeneration promoting effect calculation formula of the motor nerve injury is as follows:
statistical analysis using T test and one-way anova, p <0.05 indicated significant differences, providing representative experimental profiles.
The results show that: the length (204 pixels) of peripheral motor nerves of zebra fish in the lead poisoning model control group is obviously smaller than that of a normal control group (228 pixels), so that the success of constructing a peripheral motor nerve injury model is prompted (p is less than 0.001); the positive control drug calcium disodium edetate zebra fish peripheral motor nerve length (215 pixels) is remarkably increased (p is less than 0.05) compared with the model control group (204 pixels), and the motor nerve regeneration promoting effect is 46%, which indicates that the calcium disodium edetate has obvious regeneration promoting effect on motor nerve injury caused by lead poisoning.
The concentration of the water-soluble administration component I is 27.8, 83.3 and 250 mu g/mL, the length of peripheral motor nerve of the zebra fish is 206, 211 and 214 pixels, compared with a model control group, p is greater than 0.05& p is less than 0.05, and the regeneration promoting effect on motor nerve injury caused by lead poisoning is 8.3 percent, 29 percent and 42 percent; the peripheral motor nerve lengths of the zebra fish with the concentrations of the two components of 27.8, 83.3 and 250 mu g/mL are respectively 215 pixels, 216 pixels and 216 pixels, and compared with a model control group, the p is less than 0.05, and the regeneration promoting effect on the motor nerve caused by lead poisoning is respectively 46 percent, 50 percent and 50 percent. The first component and the second component have obvious regeneration promoting effect on peripheral motor nerve injury caused by lead poisoning. See table 3, fig. 2, fig. 3 and fig. 4 for details.
Table 3 regeneration-promoting action of peripheral motor nerve injury of zebra fish (n ═ 10) with component one and component two
P <0.05 compared to model control group
Example 5
Evaluation of the protective Effect of component one and component two on axonal injury caused by lead poisoning
240 tail 2hpf transgenic wild type AB strain zebra fish is randomly selected to be placed in a six-well plate, 30 tails are placed in each well, the volume of each well is 3mL, the concentration of a first component 27.8, 83.3 and 250 mug/mL and the concentration of a second component 27.8, 83.3 and 250 mug/mL are given to the zebra fish in a water-soluble mode, 125 ng/tail of calcium disodium edetate is given to the positive control drug in an injection mode, and meanwhile a normal control group (zebra fish treated by water for fish culture) and a model control group are set. Dissolving lead acetate in water to establish a lead poisoning model, dyeing the model by using an axon specific antibody after 70 hours of administration, taking a picture under a fluorescence microscope, collecting the fluorescence intensity (S) of the axon at the head of the zebra fish, and evaluating the axon protection effect of the pure peptide and mung bean peptide compound according to the statistical significance of the fluorescence intensity of the axon at the head. The protective effect calculation formula of the axonal injury is as follows:
statistical analysis using T test and one-way anova, p <0.05 indicated significant differences, providing representative experimental profiles. 124372
The result shows that the fluorescence intensity (707955 pixels) of the head axon of the zebra fish in the lead poisoning model control group is obviously weaker than that of the normal control group (832327 pixels), and the successful construction of the head axon injury model is prompted (p is less than 0.001). The positive control drug of the edetate calcium sodium zebra fish head axon fluorescence intensity (813117 pixels) is obviously enhanced (p is less than 0.05) compared with a model control group (707955 pixels), and the protective effect on axon injury is 85%, which shows that the edetate calcium sodium has an obvious protective effect on axon injury caused by lead poisoning.
The fluorescence intensities of the first component 27.8, 83.3 and 250 mu g/mL zebra fish head axons are 814069, 805023 and 824010 pixels respectively, compared with a model control group, p is less than 0.01 and less than or equal to 0.001, and the protection effect on axon damage caused by lead poisoning is 85%, 78% and 93% respectively. The fluorescence intensity of the zebra fish head axon with the concentration of 27.8, 83.3 and 250 mu g/mL of the second component is 813960, 829757 and 833368, and compared with the model control group, the p is less than 0.01, and the protection effect on axon damage caused by lead poisoning is 85%, 98% and 101%; the pure peptide and mung bean peptide compound has obvious protective effect on axonal injury caused by lead poisoning. See table 4, fig. 5 and fig. 6 for details.
Table 4 protective effect of zebrafish axonal injury with one component and two components (n ═ 10)
P <0.05, p <0.01, p ≦ 0.001 for the model control group
The above examples show that the mung bean peptide and the active peptide provided by the invention have the effects of promoting lead excretion, promoting regeneration of motor nerve injury caused by lead poisoning and protecting axonal injury caused by lead poisoning.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Zhongshidu Qing (Shandong) Biotechnology Co., Ltd
Application of mung bean peptide in preparation of medicine for promoting lead excretion, active peptide and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Tyr His Asp Ile Phe Met Pro Tyr
1 5
Claims (4)
1. An active peptide, which is characterized in that the amino acid sequence of the active peptide is shown as SEQ ID No. 1.
2. Use of the active peptide of claim 1 for the preparation of a medicament for promoting lead excretion.
3. Use of the active peptide of claim 1 for the manufacture of a medicament for promoting regeneration of motor nerve damage caused by lead poisoning.
4. Use of the active peptide of claim 1 for the manufacture of a medicament for protecting axonal damage caused by lead poisoning.
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